The present invention relates to a process for the fermentative preparation of amino acids such as L-lysine or L-threonine in which a strain of the bacterial genus Brevibacterium or Corynebacterium producing one or more amino acids is cultivated in a nutrient medium, and the amino acids produced are isolated from the culture fluid at the end of fermentation.
L-lysine is an essential amino acid and is used in large quantities as animal feed supplement. Numerous amino acids are generally produced biosynthetically which has long been known in the art. The bacterial strains for producing amino acids are distinguished by their capacity for secreting these amino acids into the culture medium at high concentrations within a short time. Feed batch processes are generally carried out to avoid high initial concentrations of substrate. Due to the very high metabolic capacity of production strains used, it is of decisive importance to carry out the fermentation process in such a manner that the maximum values of oxygen requirement and of evolution of heat will be of an economically acceptable order of magnitude. Various strategies have therefore been employed to regulate the metabolic activity of the organisms so as to ensure the supply of oxygen and removal of heat and at the same time balance the distribution of formation of biomass and of product.
A process entailing intermittent feeding is disclosed in CSFR-PS 212 558 in which the metabolic activity during the growth phase is adjusted by changes in pH and the total amount of biomass is adjusted by the .alpha.-aminonitrogen. Soviet Patent 157 059 describes a process entailing intermittent feeding, in which the threonine concentration serves as the criterion for the feeding and the proportion of the reducing compound is maintained at 3 to 5%. A very finely adjusted process is disclosed in French Patent 8303487. In this process, two feed solutions are continuously added: a leucine phosphate solution which is added at such a rate that both the intensity of metabolism and the formation of the biomass are limited by the rate of addition of supplement. The second feed solution, a sugar solution, is supplied at such a rate that the actual sugar concentration is maintained at 5 to 15 g/l. This process shows that due to a limitation by the leucine/phosphate supplements during the feed phase, the culture uses less sugar at any point in time than is available in the culture medium. This procedure is in line with the repeatedly documented view that both carbon-limitation (C-limitation) and undue carbon-excess (C-excess) should be avoided (e.g., East German Patent 269 167). Hadj Sassi et al. in "Biotechn. Letters," Volume 10, No. 8, pages 583-586 (1988) even propose from 90 to 140 g/l of glucose for this purpose. The metabolic activity is therefore always regulated by a factor other than that of the source of carbon.
Taxonomic Status of Corynebacterium glutamicum
Corynebacterium glutamicum ATCC 13032 (DSM 20300, IFO 12168, JCM 1318, NCIB 10025) was isolated by Kinoshita, 1957, from sewage and has been described by Kinoshita et al., 1958 as Micrococcus glutamicum. Later the strain was transferred to the genus Corynebacterium by Abe et al., 1967. Today Corynebacterium glutamicum (Kinoshita, Nakayama and Akita, 1958) (Abe, Takayama and Kinoshita, 1967) is found in the Approved Lists of Bacterial Names in the genus Corynebacterium, strain ATCC 13032 is designated as type strain of this species.
Corynebacterium glutamicum and some other coryneform bacteria are listed by Kinoshita, 1985, among the L-glutamic acid producing bacteria. Many strains of this species are found in U.S. Patent Bibliographic Database, 1997, or listed in the ATCC and DSMZ catalogues of strains as patent strains. Recently two other glutamic acid producing validly described coryneform bacteria have been classified to C. glutamicum i.e. ATCC 15990.sup.T Corynebacterium lilium, Lee and Good, 1963, and ATCC 14020.sup.T Brevibacteriurn divaricatum, Su and Yamada 1960, Corynebacterium lilium ATCC 15990 has been isolated by Lee and Good, 1963, and was officially described by Yamada and Komagata, 1972, whereas Brevibacterium divaricatum ATCC .sub.14020.sup.T was isolated and described by Sue and Yamada, 1960. Both names were listed among the validly described bacteria in the Approved List of Bacterial Names.
Suzuki and coworker (1981) found in their DNA-DNA pairing studies on glutamic acid producing corynebacteria that C. lilium, ATCC 15990.sup.T and C. glutarniculn, ATCC 13032.sup.T showed 80% DNA-DNA homology to each other. Based on the results of Suzuki and their own studies Liebl and coworker (1991) transferred Corynebacterium lilium (DSM 20137=ATCC 15990.sup.T) and B. divaricatum (DSM 20297=ATCC 14020.sup.T) as subjective synonyms to the species Corynebacterium glutamicum. In the same study three unclassified brevibacteria strains i.e. "Brevibacterium flavum" (DSM 20411=ATCC 14067), "B. lactofermentum" (DSM 1312=ATCC 13869) and (DSM 20142=ATCC 13655) were studied too. All three strains could be classified to C. glutamicum on their high DNA/DNA homology to this species. In 1987, D. Collins transferred Brevibacterium ammoniagenes (Cooke and Keith) to the genus Corynebacterium as C. ainmoniagenes (ATCC 6871). In addition, two wrongly classified Corynebacterium ammoniagenes strains i.e. ATCC 13745 and ATCC 13746 could be classified to C. glutamicum in this study too.
The classification of Micrococcus glutamicum to Corynebacterium glutamicum and the transfer of many other wrongly classified glutamic acid producing coryneform bacteria by DNA/DNA reassociation studies to this species was later confirmed and emended by Kampfer et al., in a study of 604 coryneform strains using 280 physiological characters for their numerical classification. The same results were obtained in a numerical analysis of fatty acid patterns of the genus Corynebacterium and related taxa by Kampfer and Kroppenstedt 1996. The phylogenetic analysis of the genus Corynebacterium based on the 16S rDNA gene sequence comparison carried out by Pascual et al. (1995) underlined the affiliation of Corynebacterium glutamicum to this genus.
Based on molecular biological studies (DNA/DNA reassociation and 16S rDNA sequencing) on chemotaxonomical studies (analysis of fatty acid patterns) and studies of phenetic markers (physiological tests) there is no doubt that in addition to the type strain of Corynebacterlium glutamicum ATCC 13032 other glutamic producing strains i.e. ATCC 15990 (C. lilium), ATCC 14020 (Brevibacterium divaricatum), ATCC 14067 ("B. flavum"), ATCC 13869 ("B. lactofermentum") and two wrongly classified strains of Corynebacterium ammoniagenes ATCC 13745 and ATCC 13746 belong to the species Corynebacterium glutamicum.